The Journal of Immunology Enhanced Effector and Memory CTL Responses Generated by Incorporation of Receptor Activator of NF-␬B (RANK)/RANK Ligand Costimulatory Molecules into Dendritic Cell Immunogens Expressing a Human Tumor-Specific Antigen1,2 Carsten Wiethe,3*†‡ Kurt Dittmar,* Tracy Doan,†‡ Werner Lindenmaier,* and Robert Tindle4†‡ The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-␬B (RANK)/ RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8؉ T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7- expressing DCs augmented E7-specific IFN-␥-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma. The Journal of Immunology, 2003, 171: 4121–4130. nitiation of T cell immunity depends on the Ag-presenting DCs present Ags to T cells via the TCR/MHC/epitope synapse capacity of mature dendritic cells (DCs).5 DCs occur in tis- and, in so doing, signal T cells for activation. This event requires I sues as immature cells and are specialized to capture and a second signal, also known as a costimulatory signal, which is process foreign or tumor Ag. After Ag capture, they mature in classically provided by ligation of CD28 on T cells with CD80 response to inflammatory stimuli characterized by up-regulation of (B7-1) or CD86 (B7-2) on DCs, but is now known to be provided MHC and costimulatory molecules (1), and migrate to the T cell by a family of B7 molecules, by the TNF superfamily, and by areas of draining lymph nodes where processed Ag is presented to cytokines (3, 4). ϩ T cells. DC-induced stimulation of CD8 CTL directed to tumor The interaction between DCs and T cells results in up-regulation epitopes is a vital component of the immune response to tumors (2). of receptor-ligand pairs of the TNF superfamily, including 4-1BB ligand (4-1BBL) (CD137L), FasL (CD95L), CD27, CD30, CD154 (CD40L), receptor activator of NF␬B (RANK)L (or TNF-related *Gesellschaft fu¨r Biotechnologische Forschung, Department of Molecular Biotech- activation-induced cytokine (TRANCE)), lymphotoxin, TNF-re- nology, Braunschweig, Germany; and †Sir Albert Sakzewski Virus Research Centre, lated apoptosis-inducing ligand, and members of the TNFR super- ‡ Royal Children’s Hospital, and Clinical Medical Virology Centre, University of family including 4-1BB (CD137), RANK, and CD40 (5, 6). Co- Queensland, Brisbane, Australia ligation of these receptor-ligand pairs induces downstream Received for publication April 10, 2003. Accepted for publication August 6, 2003. signaling events via TNFR-associated factor adaptor molecules. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance This signaling up-regulates adhesion and costimulatory molecules with 18 U.S.C. Section 1734 solely to indicate this fact. (7), enhances stable cellular interactions between DCs and T cells, 1 This work was supported by the National Health and Medical Research Council regulates survival of either the APC or the T cell (8), and leads to (Australia). C.W. was supported by a grant from the Helmholtz-Gemeinschaft Fors- production of T cell-stimulatory cytokines (e.g., IL-12, IL-1, and chungszentren-Strategiefond I Infektionsabwehr und Krebspra¨vention and by a short- term Ph.D. fellowship from the Deutscher Akademischer Austauschdienst. IL-6) (5, 8–10) and/or down-regulation of T cell-inhibitory cyto- 2 This paper is contribution number 192 of the Sir Albert Sakzewski Virus Research kines (e.g., IL-10) (9). A result is amplification and sustenance of Center. the ensuing immune response. 3 Current address: Dermatologische Klinik, Universitat Erlangen, Hartmannstrasse Manipulation of DCs ex vivo to display tumor epitopes before 14, D-19052 Erlangen, Germany. reinfusion is an attractive approach to tumor immunotherapy, but 4 Address correspondence and reprint requests to Dr. Robert Tindle, Sir Albert Sak- the approach is limited by the short life span of fully differentiated zewski Virus Research Centre, Royal Children’s Hospital, Herston Road, Herston, QLD 4029, Australia. E-mail address: [email protected] or mature DCs (11). This short life span is related to their rapid 5 Abbreviations used in this paper: DC, dendritic cell; RANK, receptor activator of apoptosis, but this can be relieved at least in tissue culture by NF-␬B; L, ligand; TRANCE, TNF-related activation-induced cytokine; HPV, human treatment of DCs with several costimulatory molecules, including papillomavirus; eGFP, enhanced green fluorescent protein; MOI, multiplicity of in- fection; Ad, adenovirus; CMVie, CMV immediate early; TAA, tumor-associated Ag; RANKL and CD40L (12). Furthermore, pretreatment of Ag-pulsed IRES, internal ribosome entry site. mature DCs with soluble RANKL in vitro enhances the number Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 4122 RANK/RANKL ENHANCES DC-TUMOR Ag IMMUNOGENICITY and persistence of Ag-presenting DCs in draining lymph nodes in fragment into the pCR-Blunt-II-TOPO (Invitrogen) resulting in pCRII-CD40 vivo (13). In addition, RANKL treatment increased Ag-specificT and pCRII-CD40L, respectively, we sequenced the inserts. In comparison to cell responses (13). GenBank sequences, we found in all our CD40 clones 1 aa substitution at position 227 (Met3Ile), and in the CD40L clones 1 aa substitution at position In the present study, we used novel adenovirus (Ad) vectors to 198 (Ile3Ser). This mutation in CD40L has been described (GenBank se- express a model tumor Ag (the E7 oncoprotein of human papillo- quence S21738) and does not affect biological function (18). The mutation in mavirus type 16 (HPV16)) and costimulatory molecules in DCs. CD40 has also been described (GenBank sequence X65453). To create the We investigated whether the immune response induced by immu- bicistronic expression cassette for either the murine CD40 or CD40L, the mouse ecotropic retrovirus receptor gene Rec-1 from the bicistronic construct nization with DCs expressing the tumor Ag would be enhanced by pGEMRec-1IRESegfp was replaced by the murine CD40 or CD40L gene from coprovision of RANK/RANKL or CD40/CD40L DC/T cell recep- pCRII-CD40 or pCRII-CD40L, resulting in pGEMCD40IRESegfp and tor-ligand pairs to the DCs. We reasoned that ligation of these pGEMCD40LIRESegfp, respectively. costimulatory molecules and their respective receptors within DCs Construction of Ad cosmids may effect an autocrine activation of individual DCs and/or allow reciprocal activation of interacting DCs. This would augment the rAds derived from human Ad type 5 were constructed using a cosmid activating signals received by DCs from T cells that themselves cloning procedure that allows direct assembly of rAds by cloning in Esch- erichia coli (W. Lindenmaier, unpublished data). Briefly, Ad cosmid pAd- express RANKL and CD40L. We demonstrate that coexpression cos45 was digested by XbaI and ClaIorSwaI and ClaI for CD40L, de- RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs in- phosphorylated, and ligated to the Psp1406 I/XbaI- or SwaI/XbaI-excised creased E7-directed effector and memory CTL responses. Simi- mono- or bicistronic expression cassettes including CMVie promoter and larly, we demonstrate that coexpression of the T cell costimulatory 3Ј regulatory sequences from pGEMegfp, pGEME7mutIRESegfp, pGEM- molecules (CD40L and RANKL) or DC costimulatory molecules RANKIRESegfp, pGEMRANKLIRESegfp, pGEMCD40IRESegfp, and pGEMCD40LIRESegfp, respectively. Packaging in vitro and transduction (CD40 and RANK) in E7-expressing DCs also increased E7-directed into E. coli DH5␣ yielded Ad cosmids with the corresponding expression effector and memory CTL responses. We show that augmentation of cassettes integrated into the E1 region. T cell responses correlated with up-regulation of CD80 and CD86 in rAds DCs by these costimulatory molecule combinations. rAds were propagated, purified, and titrated as described (19) with minor modifications. For production of rAd, cosmid DNA was transfected into Materials and Methods ϩ Cell lines and cell culture 293LP cells. The formation of rAds was confirmed by monitoring eGFP adenoviral plaques, and virus particles were harvested. Restriction analysis 293LP cells, an Ad-transformed human embryonic kidney cell line that of viral DNA was performed using DNA extracted from benzonase provides phenotypic complementation of the E1 genes, were purchased (Merck, West Point, PA)-digested lysates of infected cells. Purified virus from Microbix Biosystems (Toronto, Ontario, Canada). A549 cells were a was isolated by two rounds of CsCl density gradient centrifugation, and gift from T. Adrian (Medizinischen Hochschule Hannover, Hannover, Ger- extensively dialyzed. The titers of the dialyzed stocks were determined by many). The E7-expressing EL4.E7 cell line was derived from EL4 cells plaque assay on the 293LP cells. (H-2b thymoma) transfected to stably express the full-length HPV16 E7 gene (14). The EL4.A2 cell line has been described (13).